Cell death plays an important role in diabetes-induced liver dysfunction. Ferroptosis is a newly defined regulated cell death caused by iron-dependent lipid peroxidation. Our previous studies have shown that high glucose and streptozotocin (STZ) cause β-cell death through ferroptosis and that ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, improves β-cell viability, islet morphology, and function. This study was aimed to examine in vivo the involvement of ferroptosis in diabetes-related pathological changes in the liver. For this purpose, male C57BL/6 mice, in which diabetes was induced with STZ (40 mg/kg/5 consecutive days), were treated with Fer-1 . It was found that in diabetic mice Fer-1 improved serum levels of ALT and triglycerides and decreased liver fibrosis, hepatocytes size, and binucleation. This improvement was due to the Fer-1-induced attenuation of ferroptotic events in the liver of diabetic mice, such as accumulation of pro-oxidative parameters , decrease in expression level/activity of antioxidative defense-related molecules , and HMGB1 translocation from nucleus into cytosol. We concluded that ferroptosis contributes to diabetes-related pathological changes in the liver and that the targeting of ferroptosis represents a promising approach in the management of diabetes-induced liver injury. Diabetes mellitus is a chronic metabolic disorder that affects many organs including the liver . DiabeteHepatocellular death is one of the most important contributing factors to diabetes-related liver pathology and progression of liver damage . TherefoFerroptosis is a regulated, iron-dependent type of cell death whose backbone makes the cysteine/glutamate exchanger (xCT)-glutathione (GSH)-glutathione peroxidase 4 (GPX4)-membrane hydroperoxide axis . As a cyHere, we aimed to reveal the involvement of ferroptosis in diabetes-induced pathological changes in the liver. For that purpose, we examined the effects of Fer-1 on ferroptosis-related parameters in the liver of mice with streptozotocin (STZ)-induced diabetes. Fer-1 is one of the most potent ferroptosis inhibitors that exhibits high efficacy as radical-trapping antioxidants, with a particularly high potency in phospholipid bilayer membranes when compared to other antioxidants . Its metp < 0.05). Body mass of the Fer-1-treated diabetic group was not significantly different from both the control and diabetic group. Furthermore, at day 21 of the experiment, the mean serum glucose level was statistically higher in diabetic animals in comparison to the control (p < 0.01). In diabetic animals treated with Fer-1, glycemia was slightly lower than in the DM group but no significant difference was noted when compared to both the DM and Ctrl group. Regarding the hepatogram parameters, a significant elevation in the serum ALT level was found in the diabetic group when compared to the control (p < 0.05). The treatment with Fer-1 decreased ALT level compared to the DM group (p < 0.05) and restored it towards the control level. TG level was increased in the DM group (p < 0.05) in comparison to the control group and was restored with Fer-1 treatment, being lower when compared with the DM group (p < 0.05). As seen in p < 0.001) in the DM group. By contrast, Fer-1 treatment decreased the fibrotic area compared to diabetic animals (p < 0.001), although it still remained increased compared with the control group (p < 0.01). There was no difference in Vv of sinusoids between the control, DM, and DM + Fer-1 groups. The analysis of the Vv of hepatocytes revealed statistical significance among the groups and a post hoc test revealed that it decreased in the DM group compared to the control (p < 0.001). Fer-1 treatment returned the Vv of hepatocytes to the control level, thus, increasing it in comparison to the DM group (p < 0.001). Histological characteristics of the liver of control and treated mice are presented in p < 0.05) (p < 0.05). Regarding hepatocytes’ size (measured as nucleated profile surface area), a statistically significant difference was noted among the groups, and an increase was found in the DM group compared to the control ( < 0.05) c. ANOVA  < 0.05) d. Post hp < 0.001), while Fer-1 treatment alleviated this effect of DM, although it still remained below the control level (p < 0.001) (p < 0.001) and an increase in the DM + Fer-1 group when compared to the DM group (p < 0.001). Additionally, statistical significance in the activity of total SOD (p < 0.05), as well as the increase for both parameters in DM + Fer-1 group compared to the DM group (p < 0.01). As expected, we have demonstrated signs of increased hepatic lipid peroxidation including the increased iron deposition, and the accumulation of lipofuscin and 4-HNE, the second product of lipid peroxidation a. While < 0.001) c. A simi< 0.001) c where sotal SOD d and levotal SOD e were alp < 0.05). Both GSH content and GPX activity in the DM + Fer-1 group returned to the control level and increased significantly compared to the DM group .Changes in the GSH content and activities of GSH-related antioxidative enzymes are summarized in DM group a. Althoup < 0.05), while Fer-1 treatment returned it to the control level, thus, increasing it in comparison to the DM group (p < 0.05). p < 0.001). This zonation difference declines in the DM group, while Fer-1 treatment returns it toward control level (p < 0.05). Comparison of nuclear Nrf2 immunopositivity in both Z1 and Z3 revealed statistically significant differences among the groups (p < 0.001). Fer-1 treatment of diabetic animals returned Nrf2 nuclear immunopositivity to control level  and even increased it (in Z1) above control and DM level (p < 0.01).Strong Nrf2 cytoplasmic and nuclear immunopositivity of hepatocytes was noted in the liver of control animals . In addie groups c. Nucleap < 0.01) and HO-1 (p < 0.01) in the DM group and significant increase after Fer-1 treatment . Additionally, lobular zonation of xCT immunopositivity was noted in both the control and DM + Fer-1 groups, since the highest immunoreactivity was observed around the portal and the centrilobular vein, i.e., in the periportal (Z1) and pericentral area (Z3). In addition to diabetes-related changes in the expression of some ferroptosis-related downstream targets presented above . Fer-1 treatment restored the percentage of HMGB1 positive nuclei to the control level, thus increasing it compared to the diabetic group (p < 0.001). Protein content of TNF-α (p < 0.001), while Fer-1 treatment restored it to the control and decreased it compared to the DM group (p < 0.001). Although the changes in the amount of IL-6 protein are in the same direction as TNF-α, they only showed a trend of increase in the DM and a trend of decrease in the diabetic group treated with Fer-1 , a common indicator of liver damage in diabetic conditions ,26,27, w2+) with H2O2. The diabetic state is characterized by the intracellular iron deposition that has been correlated with the diabetes-induced pathological changes in many tissues, including liver [Oxidative stress is the crossing link between diabetes-induced systemic metabolic disturbances  and pathological changes, functional and morphological, in many tissues/organs, including liver . An incrng liver . Since ing liver .Along with those parameters of lipid peroxidation, we also found that Fer-1 induced restitution of diabetes-induced decrease in the level of pACC. ACC is a central enzyme involved in fatty acid biosynthesis, including long chain PUFAs and, thus, plays a context-dependent role in promoting ferroptosis. It has been shown that the inhibition of ACC blocks both erastin- and ras-selective lethal small molecule 3 (RSL3)-induced ferroptosis in mouse embryonic fibroblasts . InhibitOn the other hand, the removal of lipid peroxides is catalyzed by a specific membrane-associated isoform of GPX, GPX4. GPX4 has been considered a primary enzymatic defense mechanism against reactive oxygen species (ROS)-mediated membrane peroxides and consequently against ferroptosis, due to its strong association with membranes and its close proximity to phospholipid peroxide substrates ,41,42. SIn addition to GPX4, Fer-1 restored the activity of GST which showed a decreasing trend in diabetes. It is well-known that GST is one of the three main 4-HNE-metabolizing enzymes, among aldehyde dehydrogenase and alcohol dehydrogenase . So, theThe main regulator of redox homeostasis and cellular defense against injury is Nrf2 ,50. AfteMoreover, here, we noticed the differential expression of Nrf2 across the liver zones. Namely, the highest level of Nrf2 expression noticed in the pericentral zones in both the control and Fer-1-treated groups could be a consequence of more hypoxic and nutrient-sparse conditions in this tissue area . The proNrf2 is the key regulator of the molecules that make the core of ferroptotic signaling—xCT/GSH/GPX4. We observed that, along with the reduced Nrf2 activation in diabetes, there was also a decrease in the content of GSH and protein expression of xCT, GCLM, and GPX4 . GCLM represents the modifier subunit of GCL, a rate-limiting enzyme for GSH synthesis. GCLM binds to the catalytic subunit (GCLC) and determines the enzyme activity and, thus, GSH level . ResultsAmong those parameters tightly related to ferroptosis, we have examined one more target of Nrf2 signaling, HO-1. The Nrf2/HO-1 signaling pathway was recently described to be involved in the antagonizing oxidative stress-related damage in multiple organs and various pathological injuries . There iAlong with these pro-oxidative events, pro-inflammatory components play an important role in the pathology of the diabetic liver. In that context, HMGB1 has been recognized as important in controlling the fate of liver cells as well as in determining the type of cell death (apoptosis or autophagy) in the progression of liver damage in diabetes . SwitchiIt seems likely that ferroptosis in the liver of diabetic animals is accompanied also with the increase in level of the inflammatory cytokines (primarily TNF-α), since Fer-1 treatment abolishes the diabetes-induced increase in the level of this commonly studied marker of inflammation in the liver . In addin = 8): diabetic (DM), diabetic + Fer-1-treated (DM + Fer-1), and control (Ctrl). Diabetes was induced using multiple low doses of STZ , which were given intraperitoneally for 5 consecutive days (day 1–5) as performed previously [Male C57BL/6 mice were housed with unlimited access to standard chow and tap water at the animal facility at the Institute for Biological Research “Sinisa Stankovic”. All experimental procedures were approved by the Ethic Committee at the Institute for Biological Research “Sinisa Stankovic” (App. No 323-37-11487/2021-05) in accordance with the Directive 2010/63/EU. The 8–10 weeks old male C57BL/6 mice were divided into three groups  liver sections were routinely stained with hematoxylin and eosin. Furthermore, AZAN trichrome staining of the liver was performed to determine the volume density (Vv) of the major hepatic tissue components  . The staxP/tP, where Px is the number of points hitting the structure and Pt is the number of total points hitting the tissue [Volume densities (Vv) were determined using Vv = e tissue . The Vv 2O2 in 0.05% DAB in PBS for 10 min), while counterstaining was carried out with hematoxylin. As a positive control, sections of the spleen were used. The sections were washed, rehydrated, mounted in dibutyl phthalate polystyrene xylene (DPX) , and examined with a Leica DMLB microscope. The cells exhibiting brown particles were considered iron positive.The intracellular presence of nonheme iron (Fe(III)) was evaluated by 3,3′-diaminobenzidine (DAB)-enhanced Pearl’s iron staining . BrieflyAfter deparaffinization and rehydration, liver sections were stained with 60% Sudan III dye solution in water (0.5 g in 100 mL of 99% isopropanol). After 15 min, slides were washed, counterstained with Mayer’s hematoxylin, mounted in DPX, and examined with a Leica DMLB microscope. The cells exhibiting dark intracellular particles were considered lipofuscin-positive, and the percentage of positive cells was determined within 50 randomly selected micrographs per experimental group. To detect the expression and localization of NF-E2-related factor 2 (Nrf2), GPX4, xCT, heme oxygenase 1 (HO-1), and HMGB1, as well the formation of 4-hydroxynonenal (4-HNE)-protein adducts in the liver, immunohistochemistry was performed. The 5 μm thick liver sections were deparaffinized by xylene and rehydrated in graded ethanol. Blocking of endogenous peroxidase activity, antigen retrieval, and blocking of nonspecific binding with normal goat serum  were performed as we had implemented previously. Samples were then incubated overnight at 4 °C with following rabbit primary antibodies: anti-4-HNE , anti-Nrf2 , anti-GPX4 , anti-HO-1 , anti-HMGB1 , all purchased from Abcam  and goat anti-xCT , purchased from Santa Cruz Biotechnology . After rinsing with PBS, the sections were incubated with appropriate secondary antibodies: goat antirabbit  and donkey antigoat  for 1 h at room temperature. The final reaction product was visualized with DAB chromogen solution . After counterstaining with hematoxylin, slides were mounted in DPX and examined with a light microscope (Leica Microsystems). To quantify the HMGB1 and GPX4 tissue immunopositivity, approximately 20 fields from micrographs at 40x objective magnification were analyzed . Micrographs were analyzed in Image J software  by the Color Deconvolution2 plugin in which images were split on hematoxylin and DAB channels. DAB channel images were analyzed by random selection of fields falling on the tissue (with the omission of large vessels). Obtained grayscale values  were divided by 1000 to get the values that will be directly proportional to immunopositivity strength. To quantify the percentage of HMGB1- and GPX4-positive nuclei, approximately 20 micrographs at 20× objective magnification were analyzed  at Image J software  The total number of nuclei was used to calculate the percentage of hepatocytes exhibiting nuclear staining. Quantification of nuclear Nrf2 staining was determined from 50 micrographs at 40× magnification . The total number of nuclei was used to calculate the percentage of hepatocytes exhibiting nuclear staining.−1 mg−1 protein. Glutathione S-transferase (GST) activity was measured by the method of Habig et al. [−1 mg−1 protein. Activity of GSH reductase (GR) was assayed by the method of Glatzle et al. [−1 mg−1 protein. The content of GSH was examined in the tissue after deproteinization with 10% sulfosalicylic acid. Total GSH was measured by the enzyme-recycling assay of Griffith [−1 g−1 tissue. Total superoxide dismutase (SOD) activity was assayed using the method described by Misra and Fridovich [−1 of protein. SOD units were defined as the amount of the enzyme inhibiting epinephrine auto-oxidation under the appropriate reaction conditions. For the examinations of the antioxidative defense system, the liver tissue was dissected out and thoroughly rinsed with saline to remove traces of blood. To measure the activities of antioxidative enzymes in the liver, a 10% homogenate of the liver, prepared in sucrose buffer , was used. The activity of GPX was determined spectrophotometrically with t-butyl hydroperoxide as a substrate  and exprg et al.  and expre et al.  and exprGriffith  and exprridovich , but at For SDS-PAGE and Western blot analysis, a 10% homogenate of liver, prepared in sucrose buffer containing protease and phosphatase inhibitors , was used as described previously . ProteinSandwich ELISA was used to measure the cytokine content in liver supernatants using MaxiSorp plates . Anti-cytokine-paired antibodies were used for cytokine detection according to the manufacturer’s instructions. The antibodies were: anti-mouse TNF-α purified rabbit monoclonal (clone EPR16803-2), anti-mouse TNF-α biotinylated rabbit monoclonal , anti-mouse IL-6 purified mouse monoclonal (clone MP5-20F3), and anti-mouse IL-6 biotinylated mouse monoclonal . The absorbance was measured using multiplate reader Synergy H1 at 450 nm, with a correction at 690 nm. Standard curves, based on known concentrations of recombinant murine TNF (Abcam) and murine IL-6 (eBioscience) were used for determination of cytokine concentrations in samples and all ELISA test samples were analyzed in duplicates.F test indicated an overall difference, a one-way analysis of variance (one-way ANOVA) was performed followed by Tukey’s multiple comparisons test. To compare Nrf2 immunopositivity of two lobular zones for each group, Student’s t test was performed. The results are presented as mean value ± statistical error (SEM). Statistical significance was set at p < 0.05.Statistical analysis was performed in GraphPad Prism software . To test data for normality, the Kolmogorov-Smirnov test was used. If the Our study revealed the ferroptotic phenotype of hepatocytes as an important part of the diabetic-induced pathological changes in the liver. Moreover, the results suggest that targeting ferroptosis represents a new, promising approach in the prevention and treatment of commonly observed liver pathologies accompanying diabetes.


CircRNAs have been reported to be related to hepatocellular carcinoma (HCC) development. Limited studies have revealed the expression profile of circRNAs in tumor and para-tumor normal samples in HCC patients. We found that circASPH was significantly increased in HCC tumor samples and that the level of circASPH was closely related to the overall survival of HCC patients. Mechanistically, circASPH could regulate the methylation of the promoter and expression of hydrocyanic oxidase 2 (HAO2) to promote HCC progression by acting as a sponge for miR-370-3p, and miR-370-3p could target DNMT3b and increase the 5mC level. In summary, our study determined that circASPH could regulate the methylation and expression of HAO2 and it could be considered an important epigenetic regulator in HCC progression. Hepatocellular carcinoma (HCC) is becoming one of the most common malignancies, with a high rate of cancer-related death worldwide, increasing the burden worldwide . HepatitCircular RNAs (circRNAs) have been identified as a new type of non-coding RNAs. CircRNAs have a covalently closed loop structure without a 5’ cap and a 3’ tail, which is different from traditional linear RNA . BecauseDNA methylation is catalyzed by two types of enzymes. In the first type, after DNA replication, DNA methyltransferase 1 (DNMT1) and its cofactor could recognize hemimethylated DNA . In the Here, two circRNA-seq databases were used to identify whether circASPH was significantly upregulated in tumor tissues in HCC patients. The expression of circASPH levels in tumor tissues was closely related to the prognosis of HCC patients. Mechanistically, circASPH could regulate the methylation process and expression of HAO2 by acting as a sponge for miR-370-3p, and miR-370-3p targeted DNMT3b to increase the 5mC level. Therefore, circASPH could be considered an important epigenetic regulator in HCC progression.In total, 181 samples were collected from consecutive patients with HCC who underwent curative resection in the Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University. Fresh human HCC and adjacent nontumor liver tissue samples were blindly collected from the cohort. Informed consent was obtained from each patient, and ethical approval was granted by the Ethics Committee of The First Affiliated Hospital of Nanjing Medical University. The information is summarized in The human HCC cell lines Huh-7, SMMC-7721, HepG2, MHCC97H, and HCCLM3 were routinely maintained in our laboratory. The normal liver cell line was THLE-2. Transfection was carried out by using Lipofectamine 3000 , and total RNA was obtained according to the manufacturer’s instructions. Si-circASPH (5’-GCAAAAGGACUUUAAAGAGAUU-3’) and miR-370-3p mimics were purchased from GenePharma .The lentiviral vectors pGMLV-SC5-shmiR-370-3p and pGMLV-SC5-shRNA-circASPH and negative sequences were purchased from GenePharma (China). The lentiviral cDNA templates of circASPH and DNMT3b were cloned into the pPB-CAG vector according to the manufacturer’s instructions. Cells were then transduced with the appropriate lentivirus.MiRNAs were extracted with the mirVana™ miRNA Isolation Kit  . Real-time PCR was performed in triplicate with the SYBR Green PCR method. All miRNA levels were normalized to the U6 small nuclear RNA level; other RNA levels were adjusted using 18S as the reference. Relative expression was analyzed by the comparative cycle threshold (Ct) method. Primer sequences are listed below : circASPH, forward: 5’- ACTGCTCCCCCTGAGGAT-3, reverse: 5’- GGGACTGCTGGCTCTGAA-3’; miR-370-3p, forward: 5’-AGACCCCGCTATGGCTCTATT-3’, reverse: 5’- TTTTGGCATAACTAAGGCCGAA -3’; U6, forward: 5’- TGTAACCAGAGAGCGGGATGT-3’, reverse: 5’-AACGCTTCACGAATTTGCGT-3’. 18SrRNA, forward: 5’-CAGCCACCCGAGATTGAGCA-3’, reverse: 5’-TAGTAGCGACGGGCGGTGTG-3’; DNMT3b, forward: 5’-AGGGAAGACTCGATCCTCGTC-3’, reverse: 5’- GTGTGTAGCTTAGCAGACTGG-3’; HAO2, forward: 5’-GCATCACGCGGGATGACAA-3’, reverse: 5’- GCGATACAAATAGGGGCACTGA-3’.The protein was extracted by using Radio-Immunoprecipitation Assay (RIPA) lysis buffer. After being centrifuged, the concentration of protein was checked by a BCA kit. Proteins were separated by Sodium Dodecyl Sulfate Polyacrylamide Gel electropheresis (SDS-PAGE) and transferred to Polyvinylidenefluoride (PVDF) membranes . DNMT3b , HAO2 , and β-actin  antibodies were incubated overnight. After the incubation of the secondary antibodies, the bands were used in Image Lab after adding a chemiluminescent substrate  to visualize. Image Lab software was used to analyze the results.For 5mC  and 5hmC  staining, the treated cells or tissues were fixed with 4% PFA and washed with PBS, then permeabilized with 0.3% Triton X-100 . For immunofluorescence, after blocking, the cells or tissues were incubated with the primary antibody  overnight at 4°C. Cells or tissues were incubated with secondary antibodies and DAPI. Images were taken under the microscope. For immunocytochemistry, 3% hydrogen peroxide was used to block the endogenous peroxidase. After being washed with PBS, the tissues were incubated with 10% goat serum and the primary antibody was added for 4°C overnight. After incubating with the secondary antibody, a DAB solution was added for color staining.The wild-type (WT) and mutant (MUT) 3′-UTR fragment sequences of circASPH and human DNMT3b were constructed into pmirGLO vectors . Each cell was seeded with a proper density in 12-well plates. Alternatively, the cells were co-transfected with plasmids and mimics. After transfection, the cells were harvested and lysed. Luciferase activity was recorded using the dual-luciferase reporter assay kit .A FISH kit was used for the FISH assay. Specific probes for circASPH and miR-370-3p were synthesized by GenePharma (China). Briefly, cells were fixed with 4% paraformaldehyde, treated with 0.5% Triton X-100 , and incubated with circASPH and miR-370-3p probes overnight. Then, cell nuclei were stained with DAPI . Images were obtained with a fluorescence microscope. The sequences of probes were: circASPH probe 5′-TCTCTCTTTAAGTCCTTTTGCTTTTTGTTC-3′ and miR-370 probe 5′-ACCAGGTTCCACCCCAGCAGGC-3′.Transfected BC cells at a density of 2,000 were seeded in 96-well plates. A CCK8 assay kit  was added and incubated with cells. Then, the absorbance was measured by a microplate reader at five time periods.Transfected HCC cells were seeded in six-well plates. A 200 μl pipette tip was used to generate a linear gap. After 24 h, we used the microscope to take pictures and measured the width (W) of the scratch wound. The rate of close distance of the wounds was calculated. All measurements were carried out three times.The cell suspension was added into Transwell chamber inserts  with Matrigel. Approximately 24 h later, cells were stained and pictures taken were used to measure invasion assays under a microscope.SMMC-7721 cells were transfected with biotinylated circASPH or miRNA-370-3p  according to the standard protocol of Lipofectamine™ 3000 . After transfection, cells were harvested and lysed. Streptavidin-conjugated magnetic beads were activated and blocked with a blocking buffer for 2 h. Then, the beads were incubated with cell lysates at 4°C for 8 h to pull down the biotin-coupled RNA complex. The level of RNA was evaluated by PCR.Methylation was detected by the MeDIP kit  by applying a non-cross-reactive 5mC antibody. In the assay of the kit, DNA is sheared and then added into a microplate well and an antibody specific to methylcytosine is then used to capture methylated DNA in the wells. Then, the specificity of enriched methylated DNA was evaluated by quantitative PCR.2)/2. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University.The HCC cell lines were transfected with shRNA-circASPH and negative control (NC). Next, HCC cell lines were collected for subcutaneous injection. BALB/c nude mice were randomly divided into two groups. Mice in the control groups received subcutaneous injection with control cells, and experimental groups received a subcutaneous administration of treated HCC cells. After 5 weeks, the imaging of mice was performed and the tumor volume and weight were examined. The tumor volume was measured and calculated as follows: V = (length × widthhttp://www.targetscan.org), miRBase (http://www.mirbase.org), and miRanda (www.microrna.org) algorithms were used to screen for the targets of miRNAs. Circbase (http://circbase.org/) was used to predict the interaction between circRNAs and miRNAs. Previously published RNA-Seq and BS-Seq datasets (GSE55752) were re-analyzed. RNA-Seq reads were trimmed using Trim Galore and mapped to the GRCh38 human genome using HISAT2. Read count quantification was performed with HTSeq-count. Differential gene expression analysis was conducted with DESeq2. BS-Seq data were mapped with Bismark, and differential methylation regions were identified by Diffbind. The distribution of DMRs was analyzed by ChiPseeker and deepTools.The TargetScan . All tests were two tailed, and p<0.05 was considered statistically significant.To identify the differentially expressed circRNAs between normal tissues and HCC tumor tissues, we used two GEO databases (GSE28274 and GSE125469) for investigation  to analyze the expression of the family of DNMT. It showed that the family of DNMT was enhanced in HCC tissues  in the HCC tumor. It indicated that the DNA methylation was enhanced in HCC tumors and was enriched in the promoter area  enzyme family, including Tet 1, Tet 2, and Tet 3. 5mC and 5hmC could also also changed to thymine or 5-hydroxymethyluracil (5-hmU), depending on an alternative pathway . In HCC,via the miR-370-3p/DNMT3b/5mC/HAO2 axis. CircASPH could be considered an important epigenetic regulator in HCC progression.In conclusion, we identified that circASPH could act like an oncogene in HCC to promote cell proliferation, migration, and invasion The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by the ethics committee of the First Affiliate Hospital of Nanjing Medical University Ethics Review Board.in vitro experiments. HZ, JX, ZX, and DS analyzed data. All authors contributed to the article and approved the submitted version.CW and ZT designed the research. HZ and JX wrote the paper. JZ, JT, SH, QZ, and FZ performed the The study was supported by grants from the National Natural Science Foundation of China (Grant no. 81972675). The work was also supported in part by the Program for Development of Innovative Research Teams in the First Affiliated Hospital of NJMU, the Priority Academic Program of Jiangsu Higher Education Institutions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


P < 0.05) and were significantly higher in this group than the levels in the PD group (P < 0.05). Compared to the PD group, VOCs in the ND group increased significantly post-delivery. Further results indicated that propionic acid may be associated with metabolic disorders in pregnant women with GDM. Epidural analgesia can effectively improve the metabolism and immune function in pregnant women with GDM.Gestational diabetes mellitus (GDM) is a metabolic disease with an increasing annual incidence. Our previous observational study found that pregnant women with gestational diabetes had mild cognitive decline, which may be related to methylglyoxal (MGO). This study aimed to investigate whether labor pain aggravates the increase in MGO and explored the protective effect of epidural analgesia on metabolism in pregnant women with GDM based on solid-phase microextraction gas chromatography/mass spectrometry (SPME/GC-MS). Pregnant women with GDM were divided into a natural birth group  and epidural analgesia group . After fasting for ≥ 10 h overnight, venous blood samples were collected pre- and post-delivery to detect MGO, interleukin-6 (IL-6), and 8-epi-prostaglandin F2 alpha (8-iso-PGF2α) by ELISA. Serum samples were analyzed for volatile organic compounds (VOCs) using SPME-GC-MS. MGO, IL-6, and 8-iso-PGF2α levels in the ND group increased significantly post-delivery ( Gestational diabetes mellitus (GDM) is the most common complication . GDM canChildbirth is a physiological process that is accompanied by pain. labor pain is an unpleasant feeling due to the contraction of the uterus and transit of the fetus. Approximately 50% of the pregnant women regard labor pain as unbearable, which can instill fear . Labor pDing et al.  showed t22.1Patients aged 18–35 years with American Society of Anesthesiologists physical status I-II were admitted to the study. Sixty pregnant women with GDM were selected and divided into either a natural delivery group  or epidural analgesia group , according to whether the patient chooses labor analgesia. The study protocol was approved by the Ethics Committee of First Affiliated Hospital of Harbin Medical University and registered with the Chinese Clinical Trial Registry (registration number: ChiCTR2000038703).Patients with pre-gestational type 1 or type 2 diabetes mellitus  were not included in the study. Patients with unnatural pregnancy or gestational periods <37 weeks or >41 weeks were excluded. Subjects taking medications, including corticosteroids, antidepressants, or antiepileptics, were also excluded. Additionally, subjects with chronic metabolic, endocrine, inflammatory diseases, cancer, drug or alcohol dependency, history of major brain abnormalities , epilepsy, or Parkinson’s disease were excluded. The psychological status of the pregnant women was assessed using the Hamilton Depression Scale. Those who scored > 7 and those who may have depression were excluded.After the informed consent of the pregnant women, first of all, the pregnant women and their fetuses were monitored for oxygen inhalation and routine monitoring. At the beginning of the first stage of labor, when the uterine contraction is regular and the uterine orifice is opened to 2~3cm, let the pregnant woman take a lateral lying position, try to lower her head and hold her knees with both hands, conduct epidural puncture at the waist L2-L3 space, slowly inject the needle, place a tube 3cm to the head after the puncture is successful, and connect the syringe to draw back the bloodless and brain free spinal fluid, then inject 3 ml of 1.5% lidocaine into the epidural cavity. The epidural catheter was excluded from entering the blood vessel or subarachnoid space by mistake. When the vital signs of pregnant women are stable and there is no abnormality in the anesthesia level, the loaded dose of drug is 6 ml by epidural injection, and the drug is 0.1% ropivacaine+0.5 μg/mL sufentanil. Continuously observe the vital signs of pregnant women, and measure and maintain the anesthesia level at the T10 level. The continuous dose of the epidural link self-control analgesia pump is 4-6 mL/h, and the self-control dose is 2 ml. The administration will be stopped after the uterine orifice of the parturient is completely opened. During delivery, the two groups of pregnant women can drink sugar water and honey water to provide energy supply for pregnant women.On the survey date, all the enrolled patients underwent routine medical history inquiries, physical examinations, and provided samples for laboratory measurements. The clinical research coordinators used a standard questionnaire to collect information on demographic characteristics and medical history. All pregnant women were instructed to maintain their usual physical activity and diet for at least three days before the survey. In order to evaluate the onset of analgesia, mothers’ pain was estimated using the Visual Analogue Score  at analgesia request and at 20 minutes after administration of the initial bolus. After overnight fasting for ≥10 h, venous blood samples were collected to detect levels of glycosylated hemoglobin (HbA1c) and blood glucose (Glu). For each participant, blood was collected (3 mL) and centrifuged. The serum was recovered. Post-delivery, visual analog scale (VAS) scores were determined and the venous blood of each woman was collected and treated as just described. Blood samples were stored in a -80°C deep cryogenic refrigerator. MGO, interleukin-6 (IL-6), and 8-epi-prostaglandin F2 alpha (8-iso-pgf2α) were detected using ELISA. All measurements were performed within 6 months of sample collection.2.2via a puncture. An extraction method for headspace extraction was adopted. The SPME fiber was inserted into evacuated 20ml glass vials and exposed to the headspace of a blood sample  for 20 min at 40°C. After the extraction and concentration of the samples, the automatic sampling device inserted the extraction head into the gas chromatography-mass spectrometry (GC-MS) injection port for analysis. The desorption of volatiles occurred in a hot GC injector at 200°C for 2 min.Post-delivery, the venous blood of the two groups was analyzed using SPME. In this study, we selected a 75 μM extraction head. The coating material was carbon molecular sieve/polydimethylsiloxane (car/PDMS). An automatic sample injector was used for heating and extraction. The liquid sample bottle was accessed 2.3All analyses were performed on a model QP2010 GC/MS (Shimadzu) equipped with a DB-5MS porous layer open tubular column . The injections were performed in splitless mode, with a splitless time of 1 min. The injector temperature was set to 200°C and the carrier gas was helium at a flow rate of 2 mL/min. The temperature in the column was maintained at 40°C for 2 min to condense the hydrocarbons. The temperature was then increased to 200°C at 70°C/min and held for 1 min. Subsequently, the temperature was ramped to 230°C at a rate of 20°C/min and maintained for 3 min. MS analyses were performed in full-scan mode with an associated m/z range of 35–200 amu. An ionization energy of 70 eV was used for each measurement, and the ion source maintained at 200°C.2.4P < 0.05 indicated statistical significance.SPSS19.0 software was used for the statistical analyses. All data were tested for normality and variance. Normally distributed data are expressed as mean ± standard deviation (mean ± SD). Continuous variables with normal distribution were compared using Student’s t-test. Variables with abnormal distribution were compared using the Mann-Whitney U test. The least significant difference method was used to make multiple comparisons among the groups. Categorical data are expressed as counts and percentages, and comparisons between groups were performed by bilateral χ 2 inspection. P-value. When VIP > 1.0, and P < 0.05, the difference variable was statistically significant .SIMCA-p +11.5 software was used for multivariate data analysis and model establishment. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used for statistical analysis. At the same time, the PLS-DA sample distribution score map was used to divide different samples into different clusters. The default seven-round cross-validation method was used, and the value of the variable importance in projection (VIP) of relevant variables in the PLS-DA model was calculated. Two hundred iterative permutation tests were conducted to verify the supervision mode and to prevent overfitting of the PLS-DA model. The nonparametric Kruskal–Wallis rank sum test was used to calculate the 33.1P > 0.05) ((P < 0.05)  Table 1.3.2P > 0.05). Compared to pre-delivery, the levels of MGO, IL-6, and 8-iso-pgf2α in the PD group post-delivery showed an upward trend, but the difference was not statistically significant. However, post-delivery, the levels of MGO, IL-6, and 8-iso-pgf2α in the ND group significantly increased (P < 0.05). These levels in the ND group post-delivery were also significantly higher than those in the PD group (P < 0.05)  Table 2.3.3Compared to the PD group, the metabolic VOCs in the ND group increased significantly post-delivery. In this study, we found 17 different metabolites Table 3.4Severe labor pain makes pregnant women vulnerable to fear and anxiety, and heightens their stress. These feelings can be exacerbated in pregnant women who develop GDM. These stressors have several adverse effects on the mother and fetus. During delivery, the body is in a state of stress due to stimulations that include pain and childbirth trauma . The immUnder physiological conditions, a variety of enzymes participate in the metabolism of MGO. A small amount of MGO in the body is not sufficient to cause toxic reactions. However, metabolic disorders in patients with GDM lead to an increase in MGO production. Simultaneously, the strong stress response caused by delivery leads to an increase in levels of reactive oxygen species, which stimulate the production of MGO . In a prUnder physiological conditions, immune changes produced by delivery have protective significance for the body . HoweverThis study mainly observed the effects of different delivery methods (ND and PD) on the metabolism of pregnant women with GDM. GC-MS was used in a novel analysis of the changes in VOCs in pregnant women with GDM post-delivery. In the two groups, we found 15 kinds of VOCs, Including 10 VOCs in the ND group and 5 VOCs in the PD group. The results revealed obvious metabolic disorders in the ND group. Ten differential substances were found in the ND group, including alpha-l-galactopyranose; 6-deoxy-, cyclic 1,2:3,4-bis(butylboronate); 1-phenyl-1-(trimethylsilyloxy)ethylene; malonic acid; bis(2-trimethylsilylethyl ester); propanoic acid; trans-beta-ocimene; trans-2-dodecen-1-ol; 1-diisopropylsilyloxycyclohexane; cycloheptane; hexanal; octanal。in vivo (in vitro that pyruvate dehydrogenase (PDH) catalyzes the condensation of saturated aldehydes with pyruvate to form acyloins. We have further shown in perfused rat heart that hexanal, presumably via PDH, is converted to 3-hydroxyoctan-2-one and that it in turn can be reduced to 2,3-octanediol  and aldehydes  were identified. Active aldehydes are mainly produced during lipid and glucose metabolism (including enzymatic and non-enzymatic pathways). The enzyme pathway is usually an aldehyde intermediate or by-product produced during glucose and lipid metabolism in vivo . This isin vivo . Similartanediol .Aldehyde metabolism disorders are involved in the occurrence and development of various diseases. α-l-Galactopyranose is a carbohydrate metabolized by hexose (glucose) and cyclic sugar. L-Galactose, also known as α-L-galactose or L-galactopyranose, belongs to the class of organic compounds known as hexoses. These are monosaccharides in which the sugar unit is a is a six-carbon containing moeity. L-Galactose is a primary metabolite. Primary metabolites are metabolically or physiologically essential metabolites. They are directly involved in an organism’s growth, development or reproduction. Based on a literature review very few articles have been published on L-Galactose. L-Galactose can be metabolized into vitamins.This may be related to the supplementary food provided to pregnant women in the ND group during delivery.Bacteroides thetaiotaomicron supports the growth of Phascolarctobacterium and the accompanying production of propionic acid through the succinic acid pathway (Phascolarctobacterium have been reported in severe depression, Alzheimer’s disease, autism, and other diseases, although the heterogeneity within the disease group was also high. Therefore, the increase in propionic acid was related to the tricarboxylic acid cycle, and the metabolism of propionic acid was also related to MGO (Veillonella metabolizes exercise-induced lactic acid into propionic acid, which improves running time, determines a natural and microbial-encoded enzyme process, and improves sports performance (Propionic acid was identified as an important component in this study. Three different biochemical pathways for propionic acid production are succinic acid, acrylate, and propylene glycol. Propionic acid is the main end-product of succinic acid fermentation. The abundance of Bacteroides and Parabacteroides, the main producers of succinic acid, increased due to a high-fat diet and were positively correlated with body weight. Succinic acid produced by  pathway . High cod to MGO Figure 2formance . The delformance .in vivo and in vitro (Six substances were found in the PD group. These included decane, heptane, and other alkanes. Alkanes were also observed in the ND group. Lipid peroxidation is a pathophysiological change in various diseases, including cancer, inflammatory diseases, atherosclerosis, and aging . Alkane in vitro . This inThis study found that both groups of pregnant women had a certain degree of stress response, but the metabolic disorder of pregnant women in the ND group was more obvious, indicating that labor pain caused an increase in oxidative stress and metabolic disorders in pregnant women with GDM. In contrast, epidural analgesia can significantly improve oxidative stress metabolites in pregnant women with GDM. This is also one of the main reasons for the changes in metabolites between the two groups.Severe labor pain activates the hypothalamus pituitary adrenal axis and sympathetic adrenal medullary axis, causing a series of neuroendocrine disorders, which also promote the secretion of glucocorticoids and catecholamines. This stress also affects the body’s immune system, thus activating inflammatory factors and oxidative stress responses, including an increase in MGO. Neuroendocrine dysfunction is one of the pathophysiological mechanisms of GDM. In the process of delivery, epidural analgesia reduces the neuroendocrine imbalance in pregnant women with GDM, adjusts the immune response, and reduces the damage caused by stress trauma to the body.There were several limitations in this study. The sample size was relatively small. Postpartum follow-up was not performed. Finally, the duration of labor was not considered. It is reported that decane, heptane, and other alkanes are found in patients with temporary respiratory syndrome . Because5The data demonstrate for the first time that propionic acid, a volatile substance, may be a potential marker of cognitive dysfunction in pregnant women with GDM aggravated by labor pain. Epidural analgesia can improve the expression of MGO/inflammatory/oxidative stress factors induced by childbirth-related pain.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the First Affiliated Hospital of Harbin Medical University. The patients/participants provided their written informed consent to participate in this study.SS designed the study, collected data, and wrote and revised the manuscript; YL and GC interpreted and analyzed the data; LG collected the data; EL designed the study. All authors contributed to the article and approved the submitted version.


Agelena labyrinthica specimens, as an N-type VGCCs ligand for this study. The effect of the omega-agatoxin-Aa2a on the glutamate-induced excitotoxicity in rats was evaluated through behavioral tests including Morris Water Maze, and Passive avoidance. The syntaxin1A (SY1A), synaptotagmin1 (SYT1), and synaptophysin (SYN) genes expression were measured via Real-Time PCR. The local expression of synaptosomal-associated protein, 25 k Da (SNAP-25) was visualized using an immunofluorescence assay for synaptic quantification. Electrophysiological amplitude of field excitatory postsynaptic potentials (fEPSPs) in the input–output and LTP curves of mossy fiber were recorded. The cresyl violet staining of hippocampus sections was performed for the groups. Our results demonstrated that the omega-agatoxin-Aa2a treatment could recover the learning, and memory impairment caused by NMDA-induced excitotoxicity in rat hippocampus.Excitotoxicity is a common pathological process in Alzheimer’s disease (AD) which is caused by the over-activity of N-Methyl-D-Aspartate receptors (NMDARs). The release of neurotransmitters depends on the activity of voltage-gated calcium channels (VGCCs). Hyper-stimulation of NMDARs can enhance the releasement of neurotransmitters through the VGCCs. This malfunction of channels can be blocked by selective and potent N-type VGCCs ligand. Under excitotoxicity condition, glutamate has negative effects on the pyramidal cells of the hippocampus, which ends in synaptic loss and elimination of these cells. These events leads to learning and memory elimination through the hippocampus circuit’s dysfunction. A suitable ligand has a high affinity to receptor or channel and is selective for its target. The bioactive small proteins of venom have these characteristics. Therefore, peptides and small proteins of animal venom are precious sources for pharmacological applications. The omega-agatoxin-Aa2a was purified, and identified from  Neurodegeneration is a symptom of central nervous system diseases like as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic lateral sclerosis (ALS). Various circumstances such as aging, trauma or compression of brain tissue, extreme stress, oxidative stress, neuro-inflammation, and excitotoxicity can induce neurodegeneration . During Alzheimer’s disease (AD) extensively increases direct  and indirect (reduced productivity) health-related costs due to its effects on both quality of life and productivity of patients themselves as well as their caregivers. Also it is projected that AD costs increases due to the growth in its prevalence and treatment costs. The societal financial burden of AD is estimated to rise by about 4.9-fold in the USA (to $1.5 trillion), 2.7-fold in Europe (to €633 billion), and 9.5-fold worldwide (to $9.1 trillion), by 2050 . ConsequExcitotoxicity is an important precondition for the neurodegeneration, and can be induced by the hyper-activation of N-Methyl-D-Aspartate receptors (NMDARs). This action has a variety of consequences on pyramidal neurons of the hippocampus such as production of high amounts of various free radicals, mitochondrial dysfunction, and activation of apoptotic factors and caspases, which leads to neuro-termination . Higher Agelena labyrinthica species is a member of Agelenidae family  was used for identification of this ligand. Behavioral tasks including the Morris water maze, and passive avoidance were performed to assess learning, and memory. The SY1A, SYT1, and SYN genes expression were measured via Real-time PCR. In addition, the amount of SNAP-25 localized protein for synaptic quantification was measured through the immunofluorescence technique in the CA3 sub-region of the hippocampus. Then, the field excitatory postsynaptic potentials (fEPSP) amplitude after LTP in the Mossy Fiber pathway were recorded for evaluation of spatial memory, and memory formation. At last, the coronal section of hippocampus and particularly CA3 sub-region were stained through cresyl violet and visualized for comparison of pyramidal neuron in the experimental groups.We could not find any direct and comprehensive study regarding the effects of N-type VGCCs on the excitotoxicity of glutamate, as a cognitive impairment All chemicals were purchased from Sigma Aldrich Company  except for the others mentioned in the text.Agelena labyrinthica was photographed with a camera. The specimens were kept under suitable conditions, humidity (60%), and temperature (25°C) and fed by crickets and mealworms. The specimens were identified using the taxonomic keys  were prepared in the laboratory with this recipe  and gently crushed with a glass stirrer for 30 min. Then, pieces of the venom gland were removed from the solution by centrifugation at 13,000 rpm for 30 min at 4°C, and the supernatant was lyophilized and stored at −70°C. Protein concentration was measured by Bradford assay with bovine serum albumin as standard protein.Female spiders were separated to extract the venom. Specimens were anesthetized with CO50, the albino mice (average weight 18–20 g) were intravenously (IV) injected with crude venom. After the injection, the animals were followed up for 1 day. The Spearman-Karber method was used to calculate the LD50 dose for the crude venom and omega-agatoxin-Aa2a protein  and heated for 10 min at 100°C. An amount of 20 μl of this solution was loaded into each well of the gel [15% Sodium Dodecyl Sulfate-Polyacrylamide Gel-Electrophoresis (SDS-PAGE)] which stained with coomassie blue dye for 45 min at 25°C. Hot water and a shaker were used to decolorize the gel. Finally, the gels were scanned by Bio-5000 Gel scanner device .® 75 pg prep grade) and run over it using FPLC (Fast Protein Liquid Chromatography) system . The column was washed with PBS Buffer. The injection volume was 1,200 μL and the flow rate was 0.7 mL/min. The fractions were observed with absorbance at 280 nm and collected in a 0.75 ml fraction. The chosen fractions (marked on the graph) were collected and injected into capillary electrophoresis and gel-electrophoresis (The lyophilized crude venom (10 mg) was resuspended in 1.5 mL of PBS buffer. The DNase (0.14 mg/ml) and RNase (0.14 mg/ml) enzymes were added to the sample and incubated for 2 h at 4°C. The clear solution was injected into a gel-filtration column  analysis was performed by Waters Alliance 2695 HPLC-Micromass Quattro micro API Mass Spectrometer. Liquid chromatography separation was performed on Atlantis T3-C18 column  at 35°C. Mobile phases were 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in Hein peak .54 adult male Wistar rats, weighing 230–250 g were taken from the animal’s nest of the Faculty of Biological Sciences and Technology, University of Isfahan. They were kept in standard cages with controlled temperature (∼25°C) and humidity (∼40%), 12 h light; 12 h dark cycle, and free access to enough food and water. The ethics committee of the University of Isfahan approved the study.The work was performed on male Wistar rats, which were spat into three groups (18 rats in each group). A small area on each rat’s skull was shaved while the head was fixed using a stereotaxic instrument  to prepare for injection into the hippocampus. The PBS buffer was used as a vehicle for omega-agatoxin-Aa2a and N-Methyl d-Aspartate (NMDA). Rats were assigned into the following groups:Control Group: Received 1 μl of PBS in the CA3 sub-region of the hippocampus twice and with an interval of 30 min.NMDA-treated group: received a single dose of NMDA  in the CA3 sub-region of the hippocampus followed by 1 μl of PBS 30 min later .Omega-agatoxin-Aa2a -treated group: received a single dose of NMDA  in the CA3 sub-region of the hippocampus followed by 1 μl of omega-agatoxin-Aa2a (2 μg/μl) 30 min later .3 sub-region of the hippocampus  was found using the Paxinos and Watson rat brain atlas  injection of phenobarbital (40 mg/kg). The phenobarbital was purchased from Martindale Pharma Company . The animals were wrapped in towels and the eyes were covered with Vaseline during the operation. Then, the CAin atlas . The injThe agents were bilaterally administered into the hippocampi using an injection needle (21 gauge) connected to a 5 μl Hamilton syringe through a polyethylene tube. The agents were slowly injected into the hippocampus area for 6 min. To avoid backflow of the fluid, the needle was slowly removed 2 min after the injection.The behavioral evaluations including the Morris water maze and Passive avoidance tasks were performed a week after the stereotaxic surgery. These tasks have assessed spatial, long-term memory, and learning. Before the behavioral tests, animals were acclimatized over 2 days in the laboratory area to be adjusted to the experimental conditions and to minimize stress.The Morris water maze test is one of the most common procedures to assess spatial memory and learning performance in rodents. The walls of the water maze room must have signs and symbols that rats can use their spatial memory to find the hidden platform in the target zone. Morris water maze task has three parts including habituation, training, and testing (it has been completely described in previous work) . In thisThe passive avoidance task was applied to assess non-conceptual communication memory and learning, as this method has already been used in neurological disorders models in small laboratory animals . The ter–Δ Δ Ct method. The mRNA levels were normalized to that of GAPDH (The mRNA extraction was performed as formerly has been described by of GAPDH .3 area were executed as formerly has been described by The rats (six in each group) were anesthetized with urethane  dissolved in 0.9% normal saline solution. The surgery and LTP recording procedures from the hippocampal CAThe brains were washed with saline and sequentially fixed in 10% formaldehyde solution and then post-fixed in 15% and 25% sucrose solutions. Two-micrometer sections were cut by using a microtome instrument. Antigen retrieval was performed enzymatically for 20 min. To reduce non-specific antibody binding, the samples were blocked with the blocking agent [10% normal goat serum  and 0.3% Triton X-100 in PBS] for 30 min at 37°C. After washing, tissue sections were incubated with mouse anti−SNAP25 monoclonal  as the primary antibody at 4°C. FITC−conjugated anti−mouse IgG  were used as secondary antibody for 2 h at room temperature. The nuclei were counterstained with DAPI . The slides were visualized using an AX70 Olympus fluorescence microscope (three in each group).Tissue coronal slides were deparaffinized two times in xylene, each for 10 min. Rehydration was performed using gradient alcohol  each time for 5 min. The brain slides were stained in cresyl violet solution  for 20 min. The dehydration was done using gradient alcohol  each time for 3 min. Tissue slides were visualized using a BX40 Olympus light microscopy (three in each group).P < 0.05 was considered a significant difference).Statistical analysis was performed using Graph Pad Prism statistics software (version 8.4.3). Statistical Data were evaluated by the D’Agostino-Pearson omnibus test to examine the normal distribution. The data collected were analyzed by one-way, and one-way repeated measures analysis of variance (ANOVA). For multiple comparisons, Tukey’s test was used. All data were shown as the mean ± standard errors of the means 7+ = 1573, (M + Na + 7H)8+ = 1,376.5. The Mass/Mass spectrum of Quadro charge ion at 1,376.5 was selected for N-terminal partial sequencing as follows: b3 = 274, b4 = 403, b5 = 516, b6 = 573, b7 = 630, b8 = 745, b9 = 848, b10 = 963, b11 = 1,020, b12 = 1,183, b13 = 1,311, b14 = 1,440, and b15 = 1,568. The results of the spectrum indicated a good consistency with the omega-agatoxin-Aa2a (Accession number: P15971) sequence , and distance moved in the target zone (P < 0.0001), when compared to the control group. NMDA treatment with administration of omega-agatoxin-Aa2a increased the time spent in the target zone (P < 0.01), and distance moved in the target zone (P < 0.01) of the maze, compared to the NMDA-treated group (P < 0.01). Also, significant differences in the time spent (P < 0.01), and distance moved (P < 0.01) in the target zone of the maze were observed between the control, and NMDA + Aga groups , in the passive avoidance task. NMDA treatment with a single dose of omega-agatoxin-Aa2a increased the latency time in the NMDA + Aga group compared to the NMDA-treated group (P < 0.05). The Control group was significantly different from the NMDA + Aga group (P < 0.0001).According to P < 0.0001). Accordingly, NMDA treatment with omega-agatoxin-Aa2a decreases the time spent in the dark chamber in the NMDA + Aga group compared to the NMDA-treated group (P < 0.0001), in the passive avoidance task. There is a significant difference between the NMDA + Aga and the control groups (P < 0.0001).As shown in P < 0.0001), SYT1 (P < 0.0001), and SYN (P < 0.0001) mRNAs expression when compared to the control group. The NMDA-treated group with a single dosage injection of omega-agatoxin-Aa2a increased SY1A (P < 0.05), SYT1 (P < 0.01), and SYN (P < 0.05) mRNAs expression with respect to the NMDA-treated group. Significant differences were observed between the two examined groups  for SY1A (P < 0.001), SYT1 (P < 0.001), and SYN (P < 0.0001).3 area of the hippocampus when compared to the control group (P < 0.001). Administration of a single dose of omega-agatoxin-Aa2a after the injection of NMDA in the NMDA-treated + Aga group remarkably increased the fEPSP amplitude after LTP induction with respect to the NMDA-treated group (P < 0.01). The fEPSP amplitude in the NMDA-treated + Aga group represented a significant difference compared to the control group (P < 0.01).According to the 3 neurons, before and after LTP induction using the high-frequency stimulation (HFS) technique in all the compared groups.3 area of the hippocampus compared to the control group (P < 0.0001). The single administration of omega-agatoxin-Aa2a after injection of NMDA distinctly enhanced the expression of SNAP-25 in the CA3 area of rat hippocampus with respect to the NMDA group (P < 0.01). The NMDA + Aga group showed a significant differences in the CA3 sub-region of the rat hippocampus compared to the control group . The single administration of omega-agatoxin-Aa2a after injection of NMDA, distinctly enhanced the living pyramidal neurons in the CA3 sub-region of the hippocampus with respect to the NMDA-treated group (P < 0.01). The NMDA-treated + ligand group showed a significant difference in the CA3 sub-region of the hippocampus compared to the control group  and evaluation of the effect of omega-agatoxin-Aa2a as an N-type VGCCs ligand.The collected results from the Morris water maze and Passive Avoidance tests showed defects in cognitive memory, and learning and memory performance. The effect of NMDA treatment appeared in the rat’s behavior. The NMDA-treated rats had a critical problem to locate the hidden platform and failed to complete this task. Plus, these rats were not able to discriminate the target zone from other zones. Therefore, this group had a serious problem with cognitive memory and learning function . Despiteociative . The men3 sub-region of the hippocampus, reduction of fEPSP after LTP induction, decrease in the rate of SNAP-25 protein, and downregulation of SY1A, SYT1, and SYN mRNAs, as well as cognitive and learning memory performance elimination. However, a single injection of omega-agatoxin-Aa2a in the NMDA-treated rats led to the prevention of consequences of excitotoxicity, through the blockage of N-type VGCCs. In addition, a single injection of omega-agatoxin-Aa2a could reverse the cognitive memory and learning impairment, upregulate SNAP-25 protein, SY1A, SYT1, and SYN mRNA levels in the CA3 sub-region of the hippocampus, enhance the fEPSP after LTP induction, and prevent pyramidal neuron death. This study can be considered as a starting point for future study on evaluation of the effect of omega-agatoxin-Aa2a on the cognitive abilities such as; pain sensitivity, sensorimotor, and locomotor ability. In addition, evaluation of the effects of bioactive small proteins on the N-type and P/Q-types VGCCs as a co-treatment can be suggested for the next research.As a conclusion, the induced excitotoxicity by NMDA resulted in pyramidal neuron death in the CAmofid@pharm.mui.ac.ir, moradmand.arachnids@gmail.com, and keimasimohammed@gmail.com.The original contributions presented in the study are included in the article/supplementary material further inquiries can be directed to the corresponding authors. Requests to access these datasets should be directed to The animal study was reviewed and approved by the animal Ethics Committee of the University of Isfahan.MK conceived the original idea. MK, MM, and MRM planned the experiments. MK, KS, MJK, MA, NE, and FE performed the experiments, data collection, analysis, and interpretation. MK and MA wrote the manuscript. MM, MRM, and MA have collaborated in presenting the research idea. MK, MM, and MRM supervised the project. All authors revised the manuscript and approved the final version of manuscript.


The COVID-19 pandemic has resulted in unprecedented changes to the life of patients with cancer. In this study, we aim to evaluate the impact of the COVID-19 pandemic on the mental health and general well-being of patients with colorectal cancer by carrying out a prospective longitudinal questionnaire. We found that around one in four participants reported symptoms of anxiety and poor well-being, with 15% at risk of moderate to severe depression. Amongst others, those who were worried that the COVID-19 pandemic would have an effect on their mental health were most at risk of anxiety, depression, and poor well-being. Screening for the mental health impact of the COVID-19 pandemic on patients is essential to allow timely action from all key stakeholders in order to avoid potentially longer-term detrimental consequences.n = 122) male, 92% (n = 198) of white ethnicity. Amongst participants who completed the screening psychometric questionnaire, 24% (n = 48/203) reported anxiety (GAD-7 ≥ 5), 15% (n = 31/204) depressive symptoms (PHQ-9 ≥ 10), 3% (n = 5/190) probable post-traumatic stress disorder (PC-PTSD-5 ≥ 4), and 31% (n = 66/213) poor well-being (WHO-5 < 50). In the subgroup  who consented to and completed a follow-up survey 6 months later, there was a significant increase in the number of participants at risk of depression . Self-reported concern about the COVID-19 pandemic impacting one’s mental health is associated with increased likelihood of anxiety, depression, and poor well-being, in respective multivariate analyses. In conclusion, screening for the mental health impact of the COVID-19 pandemic is essential to ensure timely action from all key stakeholders and to avoid potentially longer-term detrimental consequences.The COVID-19 pandemic has resulted in unprecedented changes to the lives of patients with cancer. To evaluate the impact of the COVID-19 pandemic on the mental health and well-being of patients with colorectal cancer, we conducted a prospective longitudinal questionnaire study at a UK tertiary cancer centre. In total, 216 participants were included: mean age 65 years, 57% ( The coronavirus disease 2019 (COVID-19) pandemic has resulted in significant changes in the management of patients diagnosed with cancer ,4. This Despite significant progress brought about by national vaccination campaigns, most countries worldwide continue to be affected by the emergence of new strains of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in significant pressures on the healthcare systems . Some vuPICO-SM is a single centre prospective survey study conducted at a large comprehensive cancer centre in North West England, UK (The Christie NHS Foundation Trust). All participants were aged ≥ 18 years with a diagnosis of colorectal cancer and who were able to fully comprehend the patient information sheet were invited to participate. Participants who lacked capacity were excluded. Participation was entirely voluntary, and no financial incentive was offered for completion of the survey. The survey is a 30-item questionnaire, including questions on basic demographics, the current status of the individual participant’s cancer, participants’ perception of their treatment, risk factors for COVID-19 infection, and the impact of the COVID-19 pandemic on their mental health. Participants were also questioned regarding coping strategies used (if any) during the pandemic. This survey was designed by a multidisciplinary group of experienced oncologists, psychologists, psychiatrists, nurse specialists, and with input from patient representatives.The primary objective of PICO-SM was to evaluate the levels of anxiety, depression, post-traumatic stress (PTSD), and general well-being amongst patients with colorectal cancer during the COVID-19 pandemic. The following validated self-reported screening tools for the presence and/or severity of each of the outcomes of interest were used: Generalized Anxiety Disorder scale (GAD-7) for anxiety , PatientPotential participants were identified through clinic list review  and were recruited in person or remotely (via telephone or video consultations) between 7 and 28 April 2021 (timepoint 1). Printed copies of the approved versions of the participant information sheet and survey were used. Participants had the option of either completing the survey onsite or return responses using a prepaid envelope by post. Consent was obtained upon return of the completed survey. Participants were asked to indicate if they agree to be contacted again on a future date, and those who consented to this were subsequently approached again 6 months after the day of the completion of the initial survey, between 7 October 2021 and 21 January 2022 (timepoint 2). Participants were also offered the option of being contacted for a Psycho-Oncology referral for additional support should their responses score above a certain threshold.t-Test or Mann–Whitney U Test were performed for continuous data as appropriate. To compare the participants’ answers between timepoints 1 and 2 of the longitudinal survey study, we conducted McNemar test or related-samples Wilcoxon signed-rank test.Descriptive analysis for all the variables was carried out using IBM SPSS Statistics for Windows, version 26  and data represented using Microsoft Excel and GraphPad Prism version 9.3.0 for Mac . Continuous variables were expressed as means ± standard deviations, and categorical variables were expressed as frequencies and percentages. Categorical data were analysed with the use of Chi-square test or Fisher’s exact test. Continuous variables were checked for deviation from normal distribution  for each comparison. Student’s p < 0.10 on univariate analyses were included in the final model, respectively. Multivariable analysis was not conducted for PTSD due to low incidence of events. Pearson bivariate correlation analysis was used to validate the association between the key outcome measures used: GAD-7, PHQ-9, PC-PTSD-5, and WHO-5. For all the analyses, a 5% significance level was set, and p values were two-tailed.Multivariate analysis was performed using a logistic regression for binary outcomes (anxiety (GAD-7 score ≥ 5), depression (PHQ-9 score ≥ 10), and poor well-being (WHO-5 < 50)), and odds ratios (OR) with 95% confidence intervals (CIs) were calculated. Variables with A total of 473 consecutive patients with colorectal cancer were identified and invited to participate in the PICO-SM study. At the end of the study enrolment window, 216 patients (response rate 45.7%) were included in the final analysis of the primary cohort (timepoint 1). One hundred and sixty-five patients were invited to participate in the longitudinal follow-up survey 6 months later, as 51 patients were either too clinically unwell to be contacted again or had died during the follow-up period of the study. A total of 95 patients (57.5%) subsequently consented to and completed the survey at timepoint 2. n = 42/216, 19.4%) participants disclosed that they have an underlying mental health condition, with anxiety  and depression  being the most prevalent (n = 67/216) of participants identified themselves as having a condition and/or comorbidity, which may put them at higher risk if they acquire COVID-19 infection   had had a test for COVID-19, with 9 (5.3%) participants disclosing that they had had a positive test (The mean age of the index cohort was 65 years and included 122 (56.5%) male participants and 198 (91.7%) of white ethnicity . One in revalent . Importarbidity) . At the ive test . Three pive test . n = 155/216, 71.8%) of the participants felt that the COVID-19 pandemic did not affect their mental health at all or only slightly (n = 48/203) were at risk of mild to severe anxiety (GAD-7 score ≥ 5), 15.2% (n = 31/204) reported moderate to severe depressive symptoms (PHQ-9 score ≥ 10), 2.6% (n = 5/190) had probable PTSD (PC-PTSD-5 score 4–5), and 31.0% (n = 66/213) had poor well-being (WHO-5 < 50) (p < 0.05) correlation between the various psychometric screening tools used in this study. There was a strong correlation between GAD-7 and PHQ-9  and WHO-5 and GAD-7  or PHQ-9 . There was a weak to moderate correlation between PC-PTSD-5 and WHO-5 , GAD-7 , or PHQ-9 .The majority (slightly . Amongst-5 < 50) . Addition = 132/216, 61.1%) of participants were not at all or only slightly concerned about contracting COVID-19, and only a minority  felt that the COVID-19 pandemic had/will have a negative impact on their cancer treatment  were more worried about their cancer rather than the risk of COVID-19 infection , knowing where to get help with dealing with side effect , and uncertainty around when treatment or tests will restart   . n = 95 patients who completed a further follow-up survey (same questionnaire) at timepoint 2. Within this longitudinal cohort, there were no differences in the proportion of participants who were at risk of poor well-being (25.3% vs. 22.1%), anxiety (17.9% vs. 20.0%), or PTSD (4.3% vs. 4.3%) (p = 0.021)   or the government  (We tracked the responses to the screening psychometric tools for the subgroup of s. 4.3%) . However= 0.021) . Paired = 0.015) . Moreove= 0.010) .n = 115/216, 53.2%), the use of humour , change in physical activity  , and avoiding thinking about the pandemic  were most commonly reported  patients reported that they were well-supported by their families and/or friends , nurse specialists , general practitioners (GP) , and community services   of the participants felt moderately to extremely well-supported by the government   . Just ovvernment . Overall, 88.9%) .p = 0.003), those whose self-reported disease status is unknown/uncertain , and those who wanted more support  (p = 0.001)  was associated with those who were worried that the COVID-19 pandemic would have an effect on their mental health  . Meanwhi= 0.001) . p = 0.033), as did using ‘distracting self’ as a coping strategy  (p = 0.039) and ‘focusing on positives’ as a coping strategy  were both negatively associated with the risk of moderate to severe depression   . On the pression . p < 0.001), and ‘change in substance intake’ as a coping mechanism  (p = 0.003) and support from friends and/or family  were inversely associated to poor well-being (Factors which were associated with poor well-being (WHO-5 < 50) on multivariate analysis included those who were worried that the COVID-19 pandemic would have an effect on their mental health  . ‘Using ll-being .p = 0.023), depression , and poor well-being   .The delivery of cancer care continues to be affected by the unrelenting surges and waves of the COVID-19 pandemic more than two years on since the declaration of a global pandemic by the World Health Organization (WHO) . NumerouThe PICO-SM study confirms some of the key findings of our previous survey study conducted during the first phase of the COVID-19 pandemic . Cancer More than one in five participants had reported symptoms of anxiety (GAD-7 score ≥ 5), similar to 2020 levels; however, in this more recent survey, the levels of moderate to severe anxiety levels had doubled (11.3% vs. 5.5% in 2020) [Further, the PICO-SM study also interrogated the wider impact of the COVID-19 pandemic on other mental health aspects of patients with colorectal cancer. We identified that 24% of participants reported symptoms of anxiety, with 15% at risk of moderate to severe depression. This is similar to levels described in a recent meta-analysis of anxiety and depression among patients with cancer . In our We also assessed patients’ general well-being using the validated WHO-5 Well-Being Index and found that 31% had poor well-being. This is similar to levels observed in the general population in a recent nationwide cross-sectional convenience-sampling questionnaire study conducted in Austria . In our The prevalence of PTSD was low (3%) among the participants in the PICO-SM study, which is significantly lower than the 21% reported in a prospective French study by Joly et al. . In contOverall, there was no specific demographic characteristics found to be related with higher levels of distress. Ethnic minorities, younger age, and female gender have been previously reported to be factors related to psychological distress . Ethnic Strong social support has been generally linked with increased psychological resilience after a global disaster . In our Despite the strengths of our prospective study, one must acknowledge the inherent limitations of a self-reported survey study. This study was conducted at a tertiary cancer centre, which may not reflect the practices in smaller centres where support services might be more limited . The response rate to this survey study (46%) was lower than expected, which may be reflective of the stresses and other priorities for patients during the COVID-19 pandemic and may therefore result in an underestimation of the overall scale of the mental health impact described here. As discussed previously, we have also noted an underrepresentation of ethnic minorities engaged in our survey. This is, however, representative of the demographic composition of the North West of England’s general population , of whom 86% are of ‘White’ ethnicity . In addition, the PICO-SM study was only open for recruitment for three weeks, and this might have further compounded on the unintentional stochastic sampling bias. Therefore, the potential mental health issues or coping strategies used by this important subgroup of patients remain poorly understood by our study. We inferred some longitudinal comparisons to a previous study conducted at our centre , and altIn conclusion, the results from the PICO-SM study show that the COVID-19 pandemic has had a significant impact on the mental health burden of patients with colorectal cancer. Corroborating with our previous work, participants who reported concerns about the COVID-19 pandemic impacting on their mental health are at greatest risk, and therefore, this may be a practical screening tool which may be easily deployed in routine clinical practice. The real significant longer-term and wider impact of the pandemic on patients receiving care during these difficult times and their families remain unknown. The implications of our findings will need to be validated in a larger cohort of patients, including in patients with other types of malignancy, and in other severe or chronic diseases. This is vital in appropriately planning and costing health care delivery to ensure that robust support services are instituted as nations continue their efforts in making recovery plans going forward.


P < 0.001 respectively). The kappa coefficients of reliability for intra-observer and interobserver agreement for the SS visibility improvement with ICME-TXI were 0.96, 1.00, 1.00 and 0.70, 0.96, 0.96 respectively. All endoscopists preferred ICME-TXI for visualizing SS mostly for all lesions. The sensitivity, specificity, and PPV (%) of ICME-TXI for VCL C4/5 were 80, 66.7, and 63.2, respectively. ICME-TXI facilitates the visibility of the SS of SNADETs and may contribute to their preoperative diagnosis.This pilot study aimed to investigate the utility of texture and color enhancement imaging (TXI) with magnified endoscopy (ME) for the preoperative diagnosis of superficial nonampullary duodenal epithelial tumors (SNADETs). We prospectively evaluated 12 SNADETs. The visibility for ME-TXI, ME with indigo carmine (ICME)—white-light imaging (WLI), ICME-TXI compared to ME-NBI (narrow-band imaging) was scored (+ 2 to − 2 ME-NBI was set as score 0) by 3 experts. Scores + 2 and + 1 were defined as improved visibility. The intra-observer and interobserver agreement for improved visibility of surface structure (SS) was evaluated. Sensitivity, specificity, and positive predictive value (PPV) for Vienna Classification (VCL) C4/5 associated with the preoperative diagnosis of ICME-TXI were analyzed. The SS visibility score of ICME-TXI was significantly higher than that of ME-NBI, ME-TXI, and ICME-WLI ( Pancreaticoduodenectomy (PD) is a standard treatment for duodenal cancers. However, PD is an invasive treatment with a mortality rate ranging from 1 to 4%3. Therefore, it is very important to accurately diagnose SNADETs to allow the less invasive management such as underwater endoscopic mucosal resection6 or endoscopic submucosal dissection10.The incidence of identified superficial nonampullary duodenal epithelial tumors (SNADETs) has gradually increased owing to advancements in endoscopic technology12. In addition, small biopsy bites for SNADETs can cause severe fibrosis, which may become later obstacles for endoscopic resection11. Nowadays, there are more studies of the utility of image-enhanced endoscopy (IEE) for SNADETs. Magnified endoscopy (ME) with narrow-band imaging (NBI) for SNADETs is useful15. Further, the accuracy of diagnosis for the Vienna Classification (VCL) C3, C4 or C3, C4/517 has been reported to range from 65.1 and 87%15, suggesting that endoscopic diagnosis is at least comparable to biopsy.The accuracy of preoperative biopsy for SNADETs is unsatisfactory, ranging from 68 to 71.6%18. We previously reported the usefulness of TXI for visualizing gastric mucosal atrophy and gastric neoplasms18. However, its utility with ME for SNADETs has not yet been clarified.In 2020, Olympus Medical Systems Corporation  introduced a new IEE system for texture and color enhancement imaging (TXI). In TXI, an image usually obtained by white-light irradiation is divided into a texture image and a base image. After enhancing the texture and correcting the color tone and brightness, the images are combinedThus, this pilot study aimed to investigate the utility of TXI with ME for the preoperative diagnosis of SNADETs.This prospective study was conducted at Chiba University Hospital (Japan) between March and July 2021. Patients diagnosed with sporadic SNADETs were prospectively enrolled in this study before diagnostic endoscopy. After endoscopic diagnosis, all patients underwent endoscopic resection or an operation. This study was reviewed and approved by the institutional review board of Chiba University School of Medicine and was registered at the University Hospital Medical Information Network (UMIN000041436). Informed consent was obtained from all participants. All methods were performed in accordance with the relevant guidelines and regulations.18.We used the CV-1500 light source equipped with a TXI system and the GIF-XZ1200 and GIF-H290Z endoscopes . There are two types of TXI: mode 1 and mode 2. In TXI mode 1, the color tone changes are more coordinated than in TXI mode 2. In this study, we used only TXI mode 1. For the structure enhanced mode, “A7” was selected for NBI and (white-light imaging) WLI, while “strong” was selected for TXI17, and the deposition of white opaque substance (WOS)19. The macroscopic findings were assessed according to the Paris endoscopic classification20. VCL endoscopic diagnosis was based on the presence of WOS, size of the lesion, and surface structure (SS) (closed- or open-loop) as described in a previous report15. Closed-loop structures were defined as oval-shaped mucosal structures that could be interpreted as connecting the starting point of the mucosa to the endpoint. Open-loop structures were defined as linear mucosal structures15. Endoscopic diagnosis with ICME-TXI was performed separated by each endoscopist.ME with WLI (ME-WLI), NBI (ME-NBI), and ME with TXI (ME-TXI) were performed for each lesion. For ME-WLI and ME-TXI, indigo carmine (IC) dye was also used . During the preoperative endoscopic diagnosis of SNADETs, the lesions were assessed for size (mm), location in the duodenum, macroscopic findings, endoscopic diagnosis according to VCLAll the tissue samples were sent for histological analysis with standard hematoxylin and eosin stain and were evaluated by gastrointestinal pathologists. The pathological diagnosis was according to the VCL, with classification into VCL C3 and C4/5. The details of VCL C4/5 are as follows; high-grade adenoma (C4.1), noninvasive carcinoma (C4.2), suspicious for invasive carcinoma (C4.3), intramucosal carcinoma (C5.1), and submucosal carcinoma (C5.2).23. The details of the score are as follows, + 2 (improved visibility remarkably), + 1 (improved visibility), 0 (unchanged visibility), − 1 (worsened visibility), − 2 (worsened visibility remarkably). Scores + 2 and + 1 were defined as improved visibility. The intra-observer and interobserver agreements for the improved visibility of SS were evaluated. For intra-observer agreement, the first and second evaluations were spaced 6 months apart. In addition, the favored modality by the examiners for the evaluation of the SS and blood vessels was assessed. The sensitivity, specificity, and positive predictive value (PPV) for VCL C4/5 associated with the preoperative diagnosis of ICME-TXI and preoperative biopsy were also investigated.The primary endpoint of this study was the visibility of the SS for SNADETs with ICME-TXI. We also assessed the visibility of the SNADETs’ blood vessels. The visibility for ME-TXI, ICME-WLI, ICME-TXI compared to ME-NBI (ME-NBI was set as score 0) was scored by three experts (an endoscopist with > 5 years of IEE experience) in five stages, a modification of previous reports24.There were no data regarding ICME-TXI for SNADETs. As this was a pilot study, 12 lesions were enrolled based on criteria from a previous reportP values < 0.05 were considered statistically significant.Baseline data were presented as mean ± standard deviation (SD). The differences in visibility scores were analyzed using Friedman's test with Bonferroni correction. Intra-observer and interobserver agreement for the visibility improvement was assessed with Cohen’s Kappa analysis. The kappa coefficient of reliability was classified as follows: 0.0–0.2 (slight agreement), 0.21–0.40 (fair agreement), 0.41–0.60, (moderate agreement), 0.61–0.80 , and 0.81–1.0 . All statistical analyses were performed using the Statistical Package for the Social Sciences software version 26 . In total, 11 patients and 12 lesions were evaluated. The characteristics of the patients and lesions are shown in Table P value of the visibility scores of SNADETs in ME-TXI, ICME-WLI, and ICME-TXI compared to ME-NBI are shown in Table P < 0.001, respectively). The visibility scores of blood vessels in ME-TXI, ICME-WLI, ICME-TXI were significantly lower than ME-NBI . In addition, the visibility score of blood vessels in ICME-TXI was significantly higher than ICME-WLI (P = 0.003).The mean visibility scores (± SD) in each modality are shown in Fig. For visualizing SS, all endoscopists preferred ICME-TXI mostly for all lesions. On the other hand, for visualizing blood vessels, all endoscopists preferred ME-NBI mostly for all lesions.The kappa coefficients of reliability for intra-observer agreements for the visibility improvement for SS with ICME-TXI were 0.96, 1.00, and 1.00. While those of interobserver agreement for the visibility improvement for SS with ICME-TXI were 0.70, 0.96, and 0.96. Intra-observer agreements for SNADETs were “almost perfect or perfect agreement”. Interobserver agreements for SNADETs were “substantial agreement” or “almost perfect or perfect agreement.”The sensitivity, specificity, and PPV (%) for VCL C4/5 associated with a preoperative diagnosis of ICME-TXI  were 80, 66.7, and 63.2, respectively. Preoperative biopsy was performed in 9 of 12 lesions. The sensitivity, specificity, and PPV (%) for VCL C4/5 in preoperative biopsy were 40.0, 75.0, and 66.7, respectively. The sensitivity of preoperative diagnosis with ICME-TXI for SNADETs tended to be higher than with preoperative biopsy.15.This study is the first to report the usefulness of ME-TXI, including ICME-TXI. Conventionally, NBI was the main modality for ME25. For instance, the gastric phenotype had dense pattern dilatation of the intervening part with ME-NBI25. Although this study did not analyze the mucin phenotype, it could be possible to distinguish mucus phenotype preoperatively by observing the SS with ICME-TXI.The visibility of the SS with ICME-WLI and ME-TXI did not differ from that of ME-NBI. But with ICME-TXI, the SS were more visible than with ME-NBI. The visibility of the SS did not improve with IC or texture enhancement of TXI alone. It is thought that accumulated IC in the concave parts of the SNADETs’ surface made the ductal structure outline stand out due to the color emphasis. This made the SS easy to recognize. The kappa coefficients of reliability for interobserver agreement for improving visibility for SS of ICME-TXI and the percentage for endoscopists’ preference for ICME-TXI regarding the recognition of the SS were high. It seems that the reproducibility is high. Clinical effects of the results in this study were considered as follows. First, better visibility of ICME-TXI for SS might allow easier preoperative diagnosis of SNADETs even for non-expert. Second, a new diagnostic system might be constructed on the basis of clearly identified SS in the future. Akazawa et al. reported that SNADETs showed distinct endoscopic features according to the mucin phenotype15, which were comparable with our results. The sensitivity of preoperative diagnosis for ICME-TXI tended to be superior to that of biopsy in this study. ICME-TXI can recognize the SS with ease, and preoperative diagnosis may be made more efficiently.In the previous report of the ME-NBI diagnosis of SNADETs referred to for the diagnosis in this study, the sensitivity, specificity for VCL C4/5 in association with the preoperative diagnosis were 90.5% and 52.4% respectively26. Therefore, ME-NBI was still superior in vascular visibility to any other modality in this study. Interestingly, visibility scores for blood vessels of ME-TXI tended to be higher than ICME-WLI. In addition, those of ICME-TXI were significantly higher than with ICME-WLI. The outline of the blood vessel is emphasized by the texture enhancement of TXI and contrast with IC as well, so the blood vessels were easier to identify than with ICME-WLI. The diagnostic method used in this study does not include evaluating blood vessels, but TXI may be useful for diagnosis including assessing the vascular structure.NBI has an absorption range wavelength that matches the blood vessels. Capillaries in the superficial mucosal layer are emphasized by the 415 nm light and are displayed in brown, whereas deeper mucosal and submucosal vessels are visualized by the 540 nm light and are displayed in cyanThis study had several limitations. First, it was a pilot study with a sample of only 12. Therefore, the results of this study only suggested the potential of ICME-TXI in the preoperative diagnosis of SNADETs. The results of visibility score of surface structures for ICME-TXI and visibility score of blood vessels for ME-NBI that show significant differences compared to other modalities clearly were considered to be credible. On the contrary, visibility score for blood vessels of ICME-TXI was need to be analyzed with larger samples. Second, the diagnostic method referred to in this study was originally for ME-NBI. A large prospective study using a specific method for TXI for the preoperative diagnosis of SNADETs is crucial for assessing the true value of ICME-TXI.In conclusion, ICME-TXI facilitates the visibility of the SS of SNADETs and may contribute to the preoperative diagnosis of SNADETs.